This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Gene silencing is a core cellular regulatory mechanism implicated in numerous human diseases including cancer. One form of silencing, conserved across eukaryotic kingdoms, involves histone H3 lysine 9 (H3K9) methylation, proteins of the HP1 family that recognize this modification, and, intriguingly, the RNAi machinery. Despite the conservation and significance of this mechanism, the fundamental questions of how this type of silencing is initiated, how RNAi promotes histone methylation, and how repression of gene expression is ultimately affected remain largely unanswered. By constructing and assaying knockouts of genes encoding 255 factors previously reported to display punctate subnuclear localization, we identified a previously unstudied protein that we have named Ers1 which is essential for RNAi-dependent silencing in S. pombe, but dispensable for RNAi-independent silencing (Rougemaille et al., 2008). The aim of this project is to address the role of Ers1 in RNAi-dependent heterochromatin formation using a combination of biochemical and genetic tools as well as high-throughput technologies. Notably, we will test the hypothesis that this protein, which displays rapid evolutionary divergence, is a recognition factor that directs RNAs to the RNAi pathway.